The overarching goal of the present proposal is to develop a mechanistic explanation of the poorly understood trafficking processes that drive basolateral membrane localization and physiologically regulate the cell surface density of two closely related channels (Kir2.3 and Kir4.1, mutations in which are associated with EAST/Sesame syndrome) in the distal nephron. The program logically builds on our recent discoveries, defining the trafficking signals in these channels and the elucidating the intracellulr sorting and retention machinery that interact with them. Specifically, we will address the following critical and timely questions: 1. How does the Golgi Export patch in Kir channels influence basolateral sorting? Unlike conventional trafficking signals, which are typically comprised of short linear peptide sequences, we discovered that residues embedded its tertiary structure dictate Golgi exit of a prototypical potassium Kir channel. This signal patch forms a recognition site for interaction with the AP1A adaptor complex, thereby marking channels for incorporation into clathrin-coated vesicles at the trans- Golgi. Here we test the hypothesis that the conserved patch signal found in Kir2.3 and Kir4.1 initiates polarized trafficking by selecting channels as cargo for inclusion into clathrin-coated vesicles. 2. How are the basolateral trafficking signals in the C- terminal region of Kir channels interpreted? Based on our published and preliminary observation, we propose that once channels are marked for inclusion into clathrin-coated vesicles, other signals direct basolateral delivery by interacting basolateral trafficking chaperone(s). 3. What is the basis for basolateral Kir channel remodeling in the renal cortical collecting duct during potassium adaptation? This aim is designed to test the hypothesis that a previously unrecognized Kir channel remodeling process, involving Kir2.3 and Kir4.1 and a basolateral PDZ retention complex, underpins the increase in the basolateral membrane conductance in potassium adaptation. By addressing these questions, the program of investigation will provide new insights into the fundamental trafficking mechanisms that control potassium secretion in health and to understand what happens when trafficking signals and trafficking machiery goes wrong in disease. PUBLIC HEALTH RELEVANCE: Potassium channels that underpin potassium balance must be precisely organized at two polarized membrane domains in the Kidney for efficient renal potassium secretion. Disruption of ion channel trafficking and surface expression can, in fact, have devastating consequences on salt and mineral balance. Despite its importance, a long-standing and fundamental question in cell biology and physiology has been how the number and location of these membrane proteins are precisely controlled. In the present proposal, we elucidate the molecular mechanisms driving membrane trafficking of these channels in health and study what may happen when these processes go awry in disease. Thus, the studies should provide novel insights into the molecular basis of renal K handling and K homeostasis in health and disease while illuminating fundamental mechanisms of membrane protein targeting in the kidney.